Amyloid precursor protein (APP) is best known for its critical role in pathogenesis of Alzheimer's disease (AD). Relevant to this application, APP is abundantly expressed in the cerebrovascular endothelium under physiological conditions. APP is also expressed in endothelium of peripheral blood vessels but apparently at significantly lower levels than in the cerebral circulation. Strong evolutionary conservation supports the concept that APP plays an important but yet poorly defined physiological role(s). Indeed, the exact role of endothelial APP in cerebrovascular/cardiovascular homeostasis is unknown. To start bridging this gap in the existing literature, we performed extensive preliminary studies in cultured human brain microvascular endothelial cells (BMECs) and mice with genetically modified expression of APP. Next generation RNA sequencing (RNA-Seq) was employed to determine differential gene expression between control human BMECs and APP-deficient BMECs. Most notably, significant down-regulation of endothelial nitric oxide synthase (eNOS) expression was detected in APP-deficient cells. Moreover, in isolated cerebral arteries derived from APP-knockout mice, endothelium-dependent relaxations were significantly impaired. Endothelial dysfunction was associated with reduced production of NO and cyclic GMP thereby providing important in vivo evidence to suggest that intact expression and processing of APP is required for normal endothelial function. Intriguingly, only cerebral blood vessels (large arteries and microvessels) were adversely affected by the loss of APP as we did not detect significant alterations in endothelium-dependent vasomotor function in the peripheral blood vessels. Metabolism of APP was significantly impaired by aging thereby increasing vulnerability of the cerebral circulation to aging-induced endothelial dysfunction. Based on these findings we formulated following central hypothesis: endothelial APP subserves an essential protective function in endothelium of the cerebral circulation. To test this hypothesis we propose three specific aims: 1) Define molecular mechanisms and signal transduction pathways responsible for cerebrovascular endothelial function of APP, 2) Identify contributions of APP to cerebrovascular homeostasis in vivo, and 3) Determine the role of APP in cerebrovascular aging. Studies proposed in this application represent the first attempt to define physiological cerebrovascular function of APP. Successful completion of the proposed studies will identify molecular mechanisms responsible for vasoprotective properties of endothelial APP. This will have immediate impact on clinical efforts to preserve and protect healthy cerebrovascular tree in patients at risk for development of cerebrovascular disease and dementia including AD.